Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO₃^?/CO₂ as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α?carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α?carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO₃^?/CO₂. Addition of exogenous HCO₃^? or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.     

Dehydroascorbate reductase and glutathione reductase play an important role in scavenging hydrogen peroxide during natural and artificial dehydration of Jatropha curcas seeds

Changes in H₂O₂ and the main antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR), in endospermic and embryonic tissues were studied in developing and artificially dried Jatropha curcas seeds. Immature seeds were desiccation-tolerant at 80 days after flowering, as they were able to germinate fully after artificial drying on silica gel had reduced their water content to 10–12% of fresh weight. In both endospermic and embryonic tissues, H₂O₂ level and, consequently, lipid peroxide content, decreased during seed development as well as after artificial dehydration of developing seeds. All examined antioxidant enzymes except DHAR showed a decrease in total activity in mature stages as compared with early stages. Expression analysis of SOD genes revealed that the decrease in total SOD activities was related to the decrease in Cu/Zn-SOD expression, while the continuous activity of SOD during maturation was related to an increase in Mn-SOD expression. Artificial drying resulted in increased SOD and DHAR activity, irrespective of the developmental stage. Our results revealed weak participation of CAT and APX in H₂O₂ scavenging, as well as no significant alterations in GR activities either during maturation or after artificial drying. Changes in SOD and GR isoenzyme patterns occurred during maturation-related drying, but not after artificial drying. These results highlight the role of ascorbate-glutathione cycle enzymes (DHAR and GR) in H₂O₂ scavenging during maturation or after artificial drying of developing J. curcas seeds.     

High salt stress in coupled and uncoupled thylakoid membranes: A comparative study

The effect of high salt concentration on photosystem II (PS II) electron transport rates and chlorophyll a fluorescence induction kinetics was investigated in coupled and uncoupled spinach thylakoid membranes. With increase in salt concentration, the rates of electron transport mediated by PS II and the F _v/F _m ratio were affected more in uncoupled thylakoids as compared to coupled thylakoid membranes. The uncoupled thylakoid membranes seemed to behave like coupled thylakoid membranes at high NaCl concentration (∼1 M). On increasing the salt concentration, the uncoupler was found to be less effective and Na⁺ probably worked as a coupling enhancer or uncoupling suppressor. We suggest that positive charge of Na⁺ mimics the function of positive charge of H⁺ in the thylakoid lumen in causing coupled state. The function of NaCl (monovalent cation) could be carried out by even lower concentration of Ca²⁺ (divalent cation) or Al³⁺ (trivalent cation). We conclude that this function of NaCl as coupling enhancer is not specific, and in general a positive charge is required for causing coupling in uncoupled thylakoid membranes. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 6, pp. 761–767.     

Reactivation of oxygen accumulation function after complete removal of manganese from the photosystem II particles

Reactivation of 02 evolution function has been studied in PS-2 particles after complete removal of Mn and water soluble 10, 17, 24, 33 kDa proteins, It has been shown that 02 evolution function in such particles can be reactivated by adding 5 μM Mn2 and 20 mM Ca2(+). Preliminary illumination of the sample is necessary to exhibit the reactivation effect of 02 evolution. The maximum value of the reactivation of 02 evolution rate is about 60% of the control. Upon illumination of the reactivated particles with flashes of 1s duration and at a frequency of 0,1 Hz, 02 evolution occurs according to the mechanism analogous to that in the initial parties of PS 2. Thus the reactivation of water oxidation and 02 evolution after complete removal of Mn and water soluble 10, 17, 24, 33 kDa proteins resulting in the suppression of 02 evolution function has been shown for the first time and it can serve as a basic approach for profound investigation of the mechanism of photosynthetic water oxidation.     

International Conference on “Photosynthesis and Hydrogen Energy Research for Sustainability-2017”

Photosynthesis Research -     

Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery

Absorption of excess light energy by the photosynthetic machinery results in the generation of reactive oxygen species (ROS), such as H2O2. We investigated the effects in vivo of ROS to clarify the nature of the damage caused by such excess light energy to the photosynthetic machinery in the cyanobacterium Synechocystis sp. PCC 6803. Treatments of cyanobacterial cells that supposedly increased intracellular concentrations of ROS apparently stimulated the photodamage to photosystem II by inhibiting the repair of the damage to photosystem II and not by accelerating the photodamage directly. This conclusion was confirmed by the effects of the mutation of genes for H2O2-scavenging enzymes on the recovery of photosystem II. Pulse labeling experiments revealed that ROS inhibited the synthesis of proteins de novo. In particular, ROS inhibited synthesis of the D1 protein, a component of the reaction center of photosystem II. Northern and western blot analyses suggested that ROS might influence the outcome of photodamage primarily via inhibition of translation of the psbA gene, which encodes the precursor to D1 protein.     

Irreversible photoinhibition of photosystem II is caused by exposure of Synechocystis cells to strong light for a prolonged period

Irreversible photoinhibition of photosystem II (PSII) occurred when Synechocystis sp. PCC 6803 cells were exposed to very strong light for a prolonged period. When wild-type cells were illuminated at 20 degrees C for 2 h with light at an intensity of 2,500 micromol photons m(-2) s(-1), the oxygen-evolving activity of PSII was almost entirely and irreversibly lost, whereas the photochemical reaction center in PSII was inactivated only reversibly. The extent of irreversible photoinhibition was enhanced at lower temperatures and by the genetically engineered rigidification of membrane lipids. Western and Northern blotting demonstrated that, after cells had undergone irreversible photoinhibition, the precursor to D1 protein in PSII was synthesized but not processed properly. These observations may suggest that exposure of Synechocystis cells to strong light results in the irreversible photoinhibition of the oxygen-evolving activity of PSII via impairment of the processing of pre-D1 and that this effect of strong light is enhanced by the rigidification of membrane lipids.     

Carbonic Anhydrase in Subchloroplast Particles of Pea Plants

The partitioning of carbonic anhydrase (CA) activity in chloroplasts isolated from 10–14-day-old pea (Pisum sativum L.) seedlings was investigated. The effect of CA inhibitors on the kinetics of chlorophyll fluorescence in photosystem II (PSII) preparations was also studied. The activity of CA was detected in fractions of soluble proteins and in the polypeptide complexes of the PSI and PSII. Isolated particles of photosystems retained a high photochemical activity similar to that of intact chloroplasts and the high level of polyunsaturated fatty acids. The association of CA with the particles of PSII (PSII-CA) was also tested by Western-blot analysis using antibodies against PSII-CA (Cah3) from Chlamydomonas reinhardtii. The PSII particles isolated with Triton X-100 (T-20) showed a higher activity of the enzyme as calculated on a protein basis than the DT-20 particles isolated with digitonin and Triton X-100. This difference seems to be related to the higher degree of nativity of the chloroplast T-20 fragments as compared to DT-20 particles. The higher level of chlorophyll per reaction center as well as the higher content of chlorophyll b and lipid fatty acids as calculated on protein basis, in particular of E-16:113 acid, which stabilizes the oligomeric structure of the light-harvesting complex of the PSII, also confirms this suggestion. The activity of CA was not detected in the DT-20 preparations treated with Tris–HCl to eliminate manganese ions. This is likely to indicate that one of the extrinsic polypeptides of PSII exhibits CA activity. Specific inhibitors of CA (acetazolamide and imidazole) inhibited the photoinduced yield of chlorophyll fluorescence (F). This might be determined by damaging the water-oxidizing system or its interaction with the PSII reaction centers. The functional role of PSII-CA for ₂-concentrating in carboxylation sites as well as its role in the coupling of light and dark reactions in chloroplasts is discussed.